Therapeutic regeneration of diseased or damaged myocardium with pluripotent stem (PS) cells depends on the ability to efficiently pre-differentiate PS cells in vitro to specialized endpoints within the coronary vascular and cardiomyocyte lineages. In addition, optimal engraftment of PS-derived cells will likely require the isolation of cells at selected developmental endpoints within these lineages. In addition, effects of factors within the transplanted environment on stem cell behavior must be considered. This Subproject addresses each of these requirements with the objective of utilizing pluripotent human embryonic stem cells (hESCs) to re-muscularize damaged myocardium. Aim 1 tests the hypothesis that human definitive endoderm (DE) induces cardiomyogenesis in pluripotent hESCs, as well as in adult (c-kit+) stem cells. After optimizing DE-induced differentiation, factors secreted by DE will be proteomically characterized to design a biochemically-defined cardiomyogenic mixture, to be evaluated using a novel luciferase-based screening assay that monitors cardiomyogenesis in hESCs in real-time. Aim 2 will isolate Nkx-2.5+/aMHC- cells, which are early in the cardiomyogenic lineage, via FACS selection or direct biochemical induction to test the hypothesis that immature 'cardiomyoblasts', in comparison with hESC-derived mature cardiomyocytes, optimally engraft and functionally regenerate infarcted myocardium. The availability of pure Nkx-2.5+/aMHC- cells (Aim 2) and mature cardiomyocytes (Aim 1) will also enable the genetic profiling of cardiomyogenic transcriptomes, which will further inform cardiomyogenic signaling and consequently growth factor application to optimally induce differentiation. Aim 3 will assess the effects of growth factors/cytokines in myocardial interstitial fluid (MIF), a non-cellular exudate isolated from ischemic myocardium, on the behavior of hESC-derived cells in the cardiomyogenic lineage. Findings from this study will significantly contribute toward elucidation of how human heart disease may be ameliorated by via cellular therapy.